1 Anal HPV genotype distribution in HIV-infected men who have sex with men 1 by geographical ...

34 Pages·2013·0.72 MB·English

JCM Accepts, published online ahead of print on 21 August 2013
J. Clin. Microbiol. doi:10.1128/JCM.01405-13
Copyright © 2013, American Society for Microbiology. All Rights Reserved.

1 Anal HPV genotype distribution in HIV-infected men who have sex with men
2 by geographical origin, age and cytological status in a Spanish cohort
3 (CoRIS-HPV)
4 Running title: HPV genotype distribution in HIV+ MSM
5 Montserrat Torresa, Cristina Gonzálezb, Jorge del Romeroc Pompeyo Vicianad,
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6 Antonio Ocampoe, Patricia Rodríguez-Fortúnezf, Mar Masiág, José Ramón Blancoh, o
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7 Joaquín Portillai, Carmen Rodríguezc, Beatriz Hernández-Novoaj, Julia del Amob*, lo
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8 Marta Ortiza*(#) On Behalf of CoRIS-HPV study group. *Both senior authors. ed

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9 Institution at which the work was performed o
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10 Centro Nacional de Microbiología, Instituto de Salud Carlos III t
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11 Ctra. Majadahonda-Pozuelo Km 2; /jc
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12 28220 Majadahonda-Madrid, Spain .a
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13 Phone: [phone]. Fax: [phone] .o
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14 Author affiliations /
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15 a. Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain. A
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16 b. Centro Nacional de Epidemiología, Instituto de Salud Carlos III, Madrid, Spain. il 8
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17 c. Centro Sanitario Sandoval-IdISSC, Madrid, Spain. 0
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18 d. Hospital Virgen del Rocío, Sevilla, Spain. b
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19 e. Hospital Xeral, Vigo, Spain. u
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20 f. Hospital Universitario de Canarias, Tenerife, Spain.
21 g. Hospital Universitario de Elche, Alicante, Spain.
22 h. Hospital San Pedro-CIBIR, La Rioja, Spain.
23 i. Hospital General Universitario de Alicante, Alicante, Spain.
24 j. Hospital Universitario Ramón y Cajal-IRYCIS.
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25 Author to whom inquiries regarding should be directed:
26 Marta Ortiz, PhD: [email protected]
27 Centro Nacional de Microbiología, Instituto de Salud Carlos III
28 Ctra. Majadahonda-Pozuelo Km 2
29 28220 Majadahonda-Madrid
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30 Phone: [phone]. Fax: [phone] o
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31 lo
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32 Abstract
33 Knowledge on Human papillomavirus (HPV) type distribution in populations at risk
34 of Anal Cancer is needed. We described the anal HPV genotypes distribution in a
35 large Spanish cohort (CoRIS-HPV) of HIV-positive Men who have sex with Men
36 (MSM) according to geographical origin, age and cytological status. Cross-
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37 sectional analysis of baseline data from 1439 HIV-infected MSM (2007-2012) was o
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38 made. Anal HPV genotyping was performed by Linear Array. Descriptive analyses lo
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39 of the subject’s characteristics, prevalence and 95% confidence intervals (CI) were ed

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40 performed. Global prevalence of HPV, HR-HPV and LR-HPV type was 95.8%, o
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41 83.0% and 72.7%, respectively. Among HR-HPV types, HPV16 was the t
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42 commonest, followed by 59, 39, 51, 18 and 52. Prevalence of multiple HR-HPV /jc
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43 infections was 58.5%. There were no differences in crude analyses between .a
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44 Spanish and Latin-American MSM for most HPV types and a peak in prevalence .o
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45 for most HPV types was seen in patients in their late thirties. Globally and by /
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46 specific HPV groups, men with abnormal anal cytologies had higher prevalence of A
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47 infection than those without. This study has the largest number of HIV-positive il 8
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48 MSM, with HPV genotype data according to cytological status. This information can 0
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49 help design anal cancer prevention strategies in HIV-positive patients. b
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50 Keywords: HPV genotypes, MSM, HIV, anal cytology u
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51 Introduction
52 Persistent infections with high risk types of Human Papillomavirus (HR-HPV) are
53 responsible for over 80% of the cases of anal cancer (AC) (1). In the last 20-30
54 years, AC incidence has increased significantly, especially in men who have sex
55 with men (MSM), and more so in those infected with HIV (2-4). Infection with any
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56 HPV type is very frequent in HIV-positive MSM, with prevalences higher than 90%, o
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57 and HR-HPV infections are present in 48.9%-94.4% of these patients (4-6). lo
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58 Likewise, infections with multiple HR-HPV types are also very common, close to ed

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59 60% (4, 6-8). Differences in prevalences of HPV, HR-HPV types and multiple HPV o
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60 infection are observed in previous studies mainly because molecular genotyping t
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61 methods used, sample size and the analysed population. /jc
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62 Anal squamous intraepithelial lesions (SIL) are precursors of AC and, in a recent .a
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63 review a high prevalence of anal SIL- up to 57.2% - has been described in patients .o
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64 infected with HIV, similar to that of 54.7% described recently in CoRIS-HPV cohort /
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65 in Spain (4, 9). A
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66 As in invasive cervical carcinoma HPV16 is causally linked to AC and in European il 8
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67 men, is detected in 87.1% of the cases; while HPV18 is reported in only 6.2% of 0
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68 them (1, 10). b
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69 Limited data are available, though, on anal HPV type distribution in HIV-positive u
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70 subjects, and more specifically in MSM. Knowledge of the specific HPV type
71 distribution in populations at high risk of AC and its precursor lesions would be very
72 useful for vaccine design as well as for establishing the attribution of the different
73 HPV types in the natural history of the infection.
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74 The objective of this work is to describe the prevalence and distribution of anal
75 HPV genotypes in HIV-positive MSM in Spain according to their geographical
76 origin, age and cytological status.
77 Methodology
78 Subjects and methods
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79 We performed a cross-sectional analysis of baseline samples and data from o
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80 CoRIS-HPV cohort. CoRIS-HPV is a cohort study within CoRIS, the cohort of the lo
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81 Spanish Network of Excellence on HIV/AIDS Research. CoRIS is an open and ed

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82 multicentre cohort of adult patients with confirmed HIV infection, and naïve to o
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83 combined Antiretroviral Therapy (cART) at study entry, established in January t
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84 2004. Patients are followed periodically according to routine practice. CoRIS /jc
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85 collects baseline and follow-up socio-demographic (age, sex, category of .a
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86 transmission of HIV, educational level, geographical origin), clinical (AIDS and non .o
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87 AIDS-defining conditions), immunological (CD4 T cell counts), virological (HIV Viral /
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88 Load), antiretroviral treatment and vital status (including cause of death) data. A
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89 Ethics approval and signed informed consent have been obtained (11). CoRIS- il 8
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90 HPV has been described elsewhere and was set up in January 2007 to study the 0
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91 epidemiology of HPV coinfection within CoRIS (6, 9). All patients from CoRIS, b
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92 whether ART-naïve or not, were invited to participate in CoRIS-HPV. Study u
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93 subjects are informed about the nature of the study and are required to sign an ad-
94 hoc informed consent. Specific ethics approval for this study has been obtained as
95 well (Reference number of ISCIII Research Ethics Committee PI-43). Besides the
96 variables aforementioned, in CoRIS-HPV sexual behaviour variables are also
97 collected (age of first sexual intercourse, number of life-time sexual partners,
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98 number of sexual partners in the preceding 12 months and frequency of
99 unprotected intercourse in the preceding 12 months). At entry in CoRIS-HPV
100 cohort, two anal samples were collected for HPV detection and liquid cytological
101 analysis.
102 HPV DNA detection and genotyping
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103 Anal samples were collected with a cytobrush and placed into Digene Specimen o
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104 Transport Medium (Qiagen, Hilden, Germany), stored at -20ºC and shipped to the lo
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105 Retroviruses and Papillomavirus Unit of the National Centre for Microbiology in ed

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106 Madrid, for testing. DNA was extracted from a 200 µl aliquot of the original anal om

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107 sample using an automatic DNA extractor (Biorobot M48 Robotic Workstation, tt
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108 Qiagen). For quality control, in each extraction run ten samples were included, one /jc
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109 negative control (PCR quality water) and one positive control (SiHa cells infected .a
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110 with HPV16). Anal HPV infection and genotyping was determined through Linear .o
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111 Array® HPV Genotyping test (Roche Molecular Systems, Inc., Pleasanton, CA, o/
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112 USA) that detect 37 HPV types. Human β- globin gene fragment detection was Ap
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113 used as internal control. The results were considered satisfactory if there were a il 8
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114 low and high β- globin levels, or at least one HPV type was detected. As Linear 1
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115 Array HPV52 probe cross reacts with HPV33, HPV35 and HPV58, in samples in y

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116 which either of these HPV types were detected an additional HPV 52 infection e
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117 cannot be excluded. Therefore in these samples a specific HPV52 PCR system
118 designed in E6 gene was performed (12). For the analysis, HPV types were
119 classified on the basis of their association with cancer using the Muñoz et al 2006
120 classification (13). HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 was
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121 considered as HR-HPV types; HPV6, 11, 40, 42, 54, 61, 70, 72, 81 and CP6108 as
122 low- risk (LR)-HPV types; HPV26, 53, 66, 68, 73 and 82 as probably high-risk
123 (PHR)-HPV types; and HPV55, 62, 64, 67, 69, 71, 83, 84 and IS39 as
124 undetermined risk (UR)-HPV types.
125 Anal liquid cytology
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126 Anal liquid cytology samples were collected by clinicians using an endocervical o
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127 brush device (Cytobrush®, Hologic Inc., and Bedford, MA, USA). The brush was lo
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128 rinsed in ThinPrep®PreservCyt® Solution (Hologic Inc.) to resuspend the cells. ed

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129 Cytological slides were obtained using a ThinPrep 2000 processor (Hologic Inc.). o
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130 Samples with poor cellularity were considered inadequate for the cytological t
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131 analysis. Cytological results were reported following the Bethesda 2001 /jc
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132 classification, also accepted for anal cytology as: negative for intraepithelial lesion, .a
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133 ASC-US (atypical squamous cells of undetermined significance), L-SIL (low-grade .o
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134 squamous intraepithelial lesion) and H-SIL (high-grade squamous intraepithelial /
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135 lesion) (14). A
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136 Definition of variables il 8
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137 The outcome variable for the current analyses was type-specific HPV prevalence. 0
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138 Separate analyses for specific HPV type groups were performed: Any HPV type; b
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139 HR-HPV types; LR-HPV types; PHR-HPV types; UR-HPV types; HR-HPV types u
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140 belonging to alpha-7 species (HPV18, 39, 45 and 59); HR-HPV types belonging
141 to alpha-9 species (HPV16, 31, 33, 35, 52 and 58); HPV types included in the
142 quadrivalent vaccine (HPV6, 11, 16 and 18); and the most frequently HR-HPV
143 types detected in cases of AC (HPV16, 18, 31 and 33) as described by De Vuyst et
144 al. (1). The independent socio-demographic variables analysed were geographical
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145 origin (categorised as Spanish, Latin-American and Others) and age (categorised
146 as ≤ 30 years, 31-35 years, 36-40 years and ≥41 years). Anal cytology results were
147 categorised as negative anal cytology and abnormal anal cytology, which included
148 AS-CUS, L-SIL and H-SIL.
149 Statistical analysis
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150 We analysed all the subjects with baseline samples for HPV included in CoRIS- ow
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151 HPV until June 2012. Descriptive analyses of the subjects’ characteristics were lo
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152 performed. Frequency distributions are presented for qualitative variables; medians d

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153 and interquartile ranges (IQR) are presented for quantitative variables with om

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154 asymmetrical distributions. We used the chi-square test for the comparison of tt
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155 qualitative variables and non-parametric tests for the comparison of medians. /jc
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156 Prevalence and 95% confidence intervals (CI) were calculated. The analyses were .a
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157 conducted using Stata 12 (StataCorp LP, College Station, TX, USA). .o
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158 Results o/
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159 Overall, 1477 patients were included in the analyses of which 1439 had a valid A
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160 HPV DNA result (38 samples with inadequate cellularity were excluded). Of these il 8
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161 1439 subjects, 1037 also had a cytology performed, 87 of which were considered 0
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162 inadequate because of poor cellularity, leaving a final sample of 950 subjects with b
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163 valid cytological result. The majority of the patients were Spaniards (69.7%), 45 % u
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164 were naïve for antiretroviral therapy, median age was 33.6 years (IQR: 28.2-40.1)
165 and there were no differences by geographical origin and age between those with
166 a valid cytological result and those without (Table 1).
167 Prevalence of HPV genotypes
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168 Global prevalence for any HPV, HR-HPV, LR-HPV, PHR-HPV and UR-HPV types
169 were 95.8% (95% CI: 94.6-96.7), 83.0% (95% CI: 80.9-84.9), 72.7% (95% CI: 70.3-
170 75.0), 57.5% (95% CI: 54.9-60.0) and 51.1% (95% CI: 48.5-53.7), respectively
171 (Figure 1). The most common HR-HPV types were HPV16 (34.7%), HPV59
172 (20.5%), HPV39 (18.8%), HPV51 (18.6%), HPV18 and HPV52 (18.1%). HPV52
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173 prevalence obtained using an additional and specific HPV type 52 PCR in samples o
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174 infected with HPV types 33, 35 and 58 was twice (18.1% vs. 9.2 %) than that lo
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175 obtained using only Linear Array. HPV6 and CP6108 were the LR-HPV types most ed

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176 frequently detected (21.6% and 20.0%, respectively). HPV53 and 66 were the most o
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177 prevalent in the PHR-HPV group (25.8% and 18.3%, respectively) and HPV 84 and t
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178 62 in the UR-HPV group (21.8% and 14.9%, respectively). /jc
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179 HPV types belonging to alpha-7 and alpha-9 species are detected in 54.6% (95% .a
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180 CI: 52.0-57.2) and 63.9% (95% CI: 61.3-66.4) of the patients. The most frequently .o
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181 HR-HPV types detected in cases of AC (HPV16, 18, 31 and 33) were present in /
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182 57.1% (95% CI: 54.5-59.7) of anal samples and 881 patients (61.2%; 95% CI: A
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183 58.7-63.8) harboured one of the HPV types 6, 11, 16 and 18. il 8
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184 Multiple HR, HR/PHR and HR/LR-HPV infections were detected in 58.5% (95% CI: 0
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185 55.9-61.1), 51.1% (95% CI: 48.5-53.8) and 63.6% (95% CI: 61.0-66.1) of all b
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186 samples. The median number of any HPV type was 5 (IQR: 3-7) and for HR-HPV u
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187 types the median number was 2 (IQR: 1-3). In 932 MSM (64.8%: 95% CI: 62.2-
188 67.2) we detected more than four HPV types and in 292 (20.3%: 95% CI: 18.2-
189 22.5) more than four HR-HPV types.
190 HPV type distribution by geographical origin
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191 Differences in specific HPV type distribution by geographical origin were analysed
192 (Figure 2), comparing patients from Spain (N=1003) vs. patients from Latin-
193 American countries (N=319). Specific HPV type prevalences were similar in both
194 Spaniards and Latin-Americans; the only statistically significant differences were
195 observed for HPV type 31 which was more common in Spaniards (17.0% vs.
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196 12.2%, p=0.040) and for HPV types 51, 62 and 71, more frequently detected in o
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197 Latin American patients (p=0.045, p=0.025 and p<0.001, respectively). lo
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198 HPV type distribution by age ed

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199 The distribution of HPV types, in the four age categories is described in Figure 3. o
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200 Overall, HR-HPV infection was not associated with age, but a statistically t
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201 significant association with age was observed for specific HR-HPV types 16, 33, 58 /jc
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202 and 59. HPV58 prevalence increased with age (p=0.004). A slight peak in the .a
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203 prevalence for the 36-40 age category was observed for HPV types 16, 18, 31, 33, .o
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204 35, 39 and 59. /
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205 For LR-HPV types, statistically significant associations with age were observed for A
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206 HPV11, 40, 54, 70 and 81, and this effect was not observed when any LR-HPV il 8
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207 infection was considered. A slight peak in the prevalence for the 36-40 age 0
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208 categories was observed for HPV types 6, 11, 40, 54, 81 and CP6108. b
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209 For PHR and UR- HPV types, a statistically significant effect of age was observed u
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210 only for HPV68 and 73.
211 Age-specific curves for HPV types according to various classifications are shown in
212 figure 4. Only infections with any of the most HR-HPV types detected in AC cases ,
213 any of the HPV types included in the quadrivalent approval vaccine and HR-HPV
214 types belonging to alpha-7 species had a statistically significantly association with
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